Human growth hormone (hGH) is secreted in the human pituitary. In its mature form it consists of 191 amino acids, has a molecular weight of about 21,500, and thus is more than three times as large as insulin. Until the advent of recombinant DNA technology, hGH could be obtained only by laborious extraction from a limited source--the pituitary glands of human cadavers. The consequent scarcity of the substance has limited its application to treatment of hypopituitary dwarfism even though it has been proposed to be effective in the treatment of burns, wound healing, dystrophy, bone knitting, diffuse gastric bleeding and pseudarthrosis. In fact, available estimates are that the amount of hGH available from tissue is adequate only to serve about 50 percent of the victims of hypopituitary dwarfism. Thus, no hGH is available for other applications.
Recently, it has been shown that hGH can be produced in a recombinant host cell, specifically E. coli in quantities which would be adequate to treat hypopituitary dwarfism and the other conditions for which it is effective. See, for example, U.S. Pat. No. 4,342,832. While this advance in the art promises relief to those who suffer the afflictions for which it offers hope of amelioration, for reasons which are set forth below, the hGH obtained using the process of U.S. Pat. No. 4,342,832 contains at least a substantial amount of hGH to which the amino acid methionine not found in native hGH is appended at the N-terminal end of the protein. While there is no evidence that this slightly different hGH will, in sensitive individuals, cause any important undesirable side reactions it is, nevertheless, structurally distinct from "mature" hGH. Hormones which differ slightly from those produced by the human body, such as various insulins obtained as tissue extracts of cattle and other animals, have been successfully used to treat human disease for many years. Nevertheless, the advent of recombinant DNA techniques have made it possible to obtain insulin of precisely the same amino acid sequence as that produced by the body. This has been hailed not only as a great scientific advance but a medical one as well since the availability of a process for making human insulin promises to reduce the risk of adverse side reactions attendant with ingestion of animal insulin to those who suffer diabetes. Therefore, notwithstanding the availability of hGH in an active form which differs only slightly from that occurring naturally, there remains a need to obtain hGH conveniently which, in its amino acid content, consists solely of the 191 amino acid sequence of the hGH produced by the pituitary. Further, the herein invention discloses the production of met-less hGH in commercially practicable amounts.
The use of recombinant DNA technology to obtain vectors for expressing heterologous DNA in a transformed microbial host is now a well established science. The first successes in this field were achieved using strains of the gram-negative bacterium E. coli such as E. coli K-12, strain 294.
The use of E. coli as a microbial host for obtaining complex heterologous polypeptides has its limitations however. Relatively small polypeptides must be obtained as a fusion protein in which the target polypeptide is expressed as part of a larger polypeptide in order to protect the small protein from degradation by the host cell. For most purposes, the small protein produced as a fusion protein must be cleaved in some way from the larger molecule to obtain a useful product.
Large foreign proteins are not degraded by the cell and can be produced directly if the gene for their direct expression, including the appropriately placed start codon, is linked to a suitable promoter gene, such as the well known lac promoter. The signal to begin translation of the mRNA coding sequence is the AUG generated from the ATG gene codon which also codes for the amino acid methionine (Met). Because prokaryotes sometimes do not remove the N-terminal Met from the resulting protein, expression of heterologous DNA under control of a bacterial promoter and in a bacterial host sometimes results in a protein whose first amino acid is methionine. Results to date, for example, with production of hGH in E. coli, have shown that the host cell has only a limited ability to cleave methionine intracellularly and there is no convenient way to do so extracellularly. Accordingly, as noted above, microbially expressed hGH by the process of U.S. Pat. No. 4,342,832 leads to a product in which at least a substantial portion of the hGH has the appended methionine group which, in some circumstances, may cause the protein to be recognized as a foreign protein when used in therapeutic applications.
Many naturally occurring proteins are initially expressed in their normal environment with an additional peptide sequence which permits the protein to pass through a cellular membrane of the cell in which it is manufactured. The additional peptide, which is cleaved in this process, is referred to as a "signal" peptide. If a heterologous gene which included the gene for a signal sequence were placed under control of a bacterial promoter and the bacterium would cleave the signal sequence intracellularly, the mature protein without an appended methionine moiety could be obtained. However, unless cleaved by the host, the signal sequence actually complicates isolation of the mature protein since extracellular cleavage is not easily accomplished.
Efforts to produce "immature" protein, i.e., the protein of interest coupled to a signal sequence, in E. coli have suggested that gram-negative bacteria such as E. coli do not effectively process this protein to cleave the signal sequence, however. A small protein preproinsulin, has been shown to be partly processed to remove the signal peptide in E. coli. However, no success at all has been obtained with large molecules such as fibroblast and leukocyte interferons. In the case of fibroblast interferon, no biologically active material was produced (Taniguchi, T. et al., Proc. Natl. Acad. Sci. USA 77, 5230-5233 (1980)). In the case of the leukocyte interferons, biologically active material was produced but was neither transported nor properly processed.